C-MYC As A Biomarker For Tumor Sensitivity To Treatment With RNA Polymerase II Inhibition

ABSTRACT

Systems and methods for determining whether a cancer patient may respond to inhibition of RNA polymerase II as a treatment for the patient are provided. Inhibition of RNA polymerase II may be by way of chemotherapy with an agent such as triptolide, an analog of triptolide, or a prodrug form of triptolide. The cancer patient may be a pancreatic cancer patient, an ovarian cancer patient, a gastric cancer patient, or an esophageal cancer patient. The patient may have any cancer in which c-Myc is over-expressed or over-amplified.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International Application No.PCT/US15/016504, which was filed on Feb. 19, 2015, and claims priorityto U.S. Provisional Patent Application No. 61/942,650, which was filedon Feb. 21, 2014. The contents of each application are incorporated byreference herein, in their entirety and for all purposes.

FIELD OF THE INVENTION

The invention relates generally to the field of cancer treatment. Moreparticularly, the invention relates to systems and methods for assessingwhether a tumor over-expresses or over-amplifies c-Myc and, if it does,inhibiting the biologic activity of RNA polymerase II (pol II) in cellsof the tumor in order to inhibit tumor growth; c-Myc over amplificationand over-expression serves as a biomarker for inhibiting tumor growth byinhibiting RNA pol II.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference, in its entirety and for all purposes, in this document.

Pancreatic ductal adenocarcinoma (PDAC) affects 44,000 individualsyearly in the United States. Almost universally, this cancer is lethal,with very limited efficacy of chemotherapy (gemcitabine, nab-paclitaxel,platinum, 5FU). Clinical trials addressing this glaring need forexpanded portfolio of anti-cancer agents active in PDAC have beenunsuccessful, and a lack of active agents has blocked progress inimproving survival rates for pancreatic cancer. Transformative newtherapies are urgently needed for this devastating malignancy, which isunderstood to be nearly universally driven by “undruggable” andinterdependent mutations in KRAS, P53 and MYC.

Oncogenic MYC is amplified in from 8% to as high as 30% of PDAC. MYCexpression is directly regulated by multiple KRAS effectors, so thatphosphorylation of Serine-62 by ERK stabilizes MYC, whereas subsequentphosphorylation of Threonine-58 by GSK3b (which is inhibited by AKT) isrequired for MYC ubiquitin-mediated degradation. In addition to MYC'sability to function as a bHLH transcriptional factor, MYC causestranscription amplification. Tumors expressing high MYC levels showincreased levels of MYC in the promoter regions of actively transcribedgenes. Frequent amplification of c-Myc in human cancers has been thefocus of investigation, but it is believed that no effective way tocurtail viability of c-Myc-dependent cancers has yet been uncovered.

Mechanistically, suppressing MYC necessitates shutting down thepre-existing oncogenic transcriptional program including the MYC geneitself. This is achievable through interference with various componentsof RNA Pol II complex. The choice of anti-MYC targets, however, remainslimited.

SUMMARY OF THE INVENTION

A system for determining the sensitivity of a tumor to RNA polymerase IIinhibition comprises a data structure comprising reference values for alevel of c-Myc, mRNA, a level of c-MYC protein, or both a level of c-MycmRNA and a level of c-MYC protein corresponding to a degree ofsensitivity of a tumor to treatment with an agent that inhibits thebiologic activity of RNA polymerase II and a processor operablyconnected to the data structure. The processor is preferably programmedto compare a level of c-Myc mRNA, a level of c-MYC protein, or a levelof both c-Myc mRNA and c-MYC protein determined from a subject with thereference values for the level of c-Myc mRNA, level of c-Myc protein, orboth the level of c-Myc mRNA and c-MYC protein in the data structure,and also programmed to determine a RNA polymerase II inhibitionsensitivity score as a result of the comparison. The system may comprisecomputer readable media comprising executable code for causing theprocessor to compare the level of c-Myc mRNA, the level of c-MYCprotein, or the level of both c-Myc mRNA and c-MYC protein determinedfrom the cell isolated from the tumor with the reference values, andexecutable code for causing the processor to determine a RNA polymeraseII inhibition sensitivity score as a result of the comparison.

The RNA polymerase II inhibition sensitivity score preferably comprisesa likelihood that the agent will or will not inhibit the biologicactivity of RNA polymerase II in the tumor, thereby treating the tumor.Sensitivity includes killing of tumor cells. The degree of sensitivitymay take into account whether the tumor cells are resistant to the agentor particular doses of the agent.

The agent may inhibit the expression or the biologic activity of theexcision repair cross-complementing rodent repair deficiency,complementation group 3 (ERCC3) protein. The agent may inhibit thetranscription of the ERCC3 gene. The agent may inhibit the translationof ERCC3 mRNA. The agent may inhibit the biologic activity of the ERCC3protein. The agent may inhibit the transcription of the c-Myc gene. Theagent may inhibit the expression of the C-MYC protein. The agent mayinhibit the interaction of the ERCC3 protein with the C-MYC protein. Theagent may inhibit the KRAS-ERK-MYC signal cascade. The agent may inducedegradation of RNA polymerase II. The agent may induce proteasomedegradation of RNA polymerase II. The agent may comprise triptolide, ananalog of triptolide, or a prodrug form of triptolide.

The tumor may comprise cells that over-amplify c-Myc mRNA. The tumor maycomprise cells that over-express the c-Myc protein. In some preferredaspects, the tumor comprises a tumor of the pancreas. A non-limitingexample of a pancreatic tumor is pancreatic ductal adenocarcinoma(PDAC). In some preferred aspects, the tumor comprise a tumor of theovaries. In some preferred aspects, the tumor comprises a tumor of thestomach. In some preferred aspects, the tumor comprises a tumor of theesophagus.

A method for treating a tumor in a subject in need thereof comprisescomparing the level of c-Myc mRNA, the level of c-MYC protein, or thelevel of both c-Myc mRNA and c-MYC protein determined from a cellisolated from the tumor with reference values for a level of c-Myc mRNA,c-MYC protein, or both c-Myc mRNA and c-MYC protein corresponding to adegree of sensitivity of a tumor to treatment with an agent thatinhibits the biologic activity of RNA polymerase II, and if the level ofc-Myc mRNA, the level of c-MYC protein, or the level of both c-Myc mRNAand c-MYC protein is greater than or equal to a level of c-Myc mRNA, alevel of c-MYC protein, or a level of both c-Myc mRNA and c-MYC proteincorresponding to a degree of sensitivity of the tumor to treatment withan agent that inhibits the biologic activity of RNA polymerase II,contacting the tumor with an agent that inhibits the biologic activityof RNA polymerase II, thereby treating the tumor. The tumor may comprisecells that over-amplify c-Myc mRNA. The tumor may comprise cells thatover-express the c-Myc protein. Preferably, the tumor comprises a tumorof the pancreas. Preferably, the tumor comprise a tumor of the ovaries.Optionally, the method may comprise isolating the cell from the tumor.Optionally, the method may comprise determining the level of c-Myc mRNA,the level of c-MYC protein, or the level of both c-Myc mRNA and c-MYCprotein

A method for treating a tumor in a subject in need thereof comprisesdetermining whether the level of c-Myc mRNA, the level of c-MYC protein,or the level of both c-Myc mRNA and c-MYC protein in the tumor isgreater than or equal to a level of c-Myc mRNA, a level of c-MYCprotein, or a level of both c-Myc mRNA and c-MYC protein correspondingto a degree of sensitivity of the tumor to treatment with an agent thatinhibits the biologic activity of RNA polymerase II, and if the level ofc-Myc mRNA, the level of c-MYC protein, or the level of both c-Myc mRNAand c-MYC protein in the tumor is greater than or equal to a level ofc-Myc mRNA, a level of c-MYC protein, or a level of both c-Myc mRNA andc-MYC protein corresponding to a degree of sensitivity of the tumor totreatment with an agent that inhibits the biologic activity of RNApolymerase II, administering to the subject an agent that inhibits thebiologic activity of RNA polymerase II in an amount effective to inhibitthe biologic activity of RNA polymerase II, thereby treating the tumor.The tumor may comprise cells that over-amplify c-Myc mRNA. The tumor maycomprise cells that over-express the c-Myc protein. Preferably, thetumor comprises a tumor of the pancreas, ovaries, stomach, or esophagus.Optionally, the method may comprise determining the level of c-Myc mRNA,the level of c-MYC protein, or the level of both c-Myc mRNA and c-MYCprotein in the tumor. Optionally, the method may comprise isolating acell from the tumor and determining the level of c-Myc mRNA, the levelof c-MYC protein, or the level of both c-Myc mRNA and c-MYC protein inthe cell.

In accordance with the methods, the agent to be administered orotherwise contacted with the tumor may inhibit the expression or thebiologic activity of the excision repair cross-complementing rodentrepair deficiency, complementation group 3 (ERCC3) protein. The agentmay inhibit the transcription of the ERCC3 gene. The agent may inhibitthe translation of ERCC3 mRNA. The agent may inhibit the biologicactivity of the ERCC3 protein. The agent may inhibit the transcriptionof the c-Myc gene. The agent may inhibit the expression of the C-MYCprotein. The agent may inhibit the interaction of the ERCC3 protein withthe C-MYC protein. The agent may inhibit the KRAS-ERK-MYC signalcascade. The agent may induce degradation of RNA polymerase II. Theagent may induce proteasome degradation of RNA polymerase II. The agentmay comprise triptolide, an analog of triptolide, or a prodrug form oftriptolide.

A method for screening a cancer patient for the likelihood of respondingpositively to an agent that inhibits the biologic activity of RNApolymerase II comprises determining the level of c-Myc mRNA, the levelof c-MYC protein, or the level of both c-Myc mRNA and c-MYC protein froma cell isolated from the patient, entering the determined level into asystem for determining the sensitivity of a tumor to RNA polymerase IIinhibition, causing the processor of the system to compare the leveldetermined from the patient with the system's reference values of alevel of c-Myc mRNA, a level of c-MYC protein, or a level of both c-MycmRNA and c-MYC protein corresponding to a degree of sensitivity of thetumor to treatment with an agent that inhibits the biologic activity ofRNA polymerase II, and determining a RNA polymerase II inhibitionsensitivity score from the comparison. The patient may have a tumorcomprising cells that over-amplify c-Myc mRNA. The patient may have atumor comprising cells that over-express the c-Myc protein. The patientmay have a tumor of the pancreas, for example, a PDAC tumor. The patientmay have a tumor of the ovaries. The patient may have a tumor of thestomach. The patient may have a tumor of the esophagus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows patient-derived pancreatic cancer xenografts in micetreated for 21. days with various chemotherapeutic agents, with limitedefficacy. FIG. 1B shows robust control of the xenograft tumors in vivo(vehicle treated mice). FIG. 1C shows complete regression ofMYC-amplified pancreatic tumors (YT037 and PNX001) in mice after 21 daysof treatment with triptolide. MYC-normal tumors (PNX015 and PNX017)showed only transient shrinkage after triptolide treatment. Each line isaveraged volumes, n=5 mice/group.

FIG. 2A shows differential sensitivity of re-programmed patient-derivedPDAC cells to transcriptional inhibition with triptolide. FIG. 2B showsrapid suppression of MYC mRNA in resistant and sensitive cells. FIG. 2Cshows persistence of p-MYC protein and high levels of RPB1-CTDphosphorylation in refractory PNX007, but not in sensitive PNX001 cells.Cells were treated overnight with triptolide, as indicated.

FIG. 3A shows resistance emergence after initial dramatic response ofPNX001 to triptolide. FIG. 3B shows resistant tumors have markedlyincreased pMYC and ERCC3.

FIG. 4A shows interdependent MYC and ERCC3 protein levels 48 hours aftersiRNA silencing. FIG. 4B shows repression with Tet in P493-6 cellsregulates ERCC3.

FIG. 5A and FIG. 5B show that ERCC3 regulates growth and confersresistance to triptolide. FIG. 5A shows HEK293 cells transfected with anERCC3 cDNA expression construct grew faster (solid lines) and resistedtriptolide (12.5 nM, dashed lines). FIG. 5B shows a micrograph of HEK293cells treated with triptolide 25 nM.

FIG. 6 shows an RTK array showing increased FGFR1 expression in PNX001explants that acquired resistance to triptolide (panel A) compared totriptolide-sensitive samples (panel B).

DETAILED DESCRIPTION OF THE INVENTION

Various terms relating to aspects of the present invention are usedthroughout the specification and claims. Such terms are to be giventheir ordinary meaning in the art, unless otherwise indicated. Otherspecifically defined terms are to be construed in a manner consistentwith the definition provided in this document.

As used throughout, the singular forms “a,” “an,” and “the” includeplural referents unless expressly stated otherwise.

Inhibiting includes, but is not limited to, interfering with, reducing,decreasing, blocking, preventing, delaying, inactivating, desensitizing,stopping, knocking down (e.g., knockdown), and/or downregulating thebiologic activity or expression of a protein or biochemical pathway.

The terms express, expressed, or expression of a nucleic acid moleculeinclude the biosynthesis of a gene product. The term encompasses thetranscription of a gene into RNA, the translation of RNA into a proteinor polypeptide, and all naturally occurring post-transcriptional andpost-translational modifications thereof.

The terms subject and patient are used interchangeably. A subject may beany animal, and preferably is a mammal. A mammalian subject may be afarm animal (e.g., sheep, horse, cow, pig), a companion animal (e.g.,cat, dog), a rodent or laboratory animal (e.g., mouse, rat, rabbit), ora non-human primate (e.g., old world monkey, new world monkey). Humanbeings are highly preferred.

It has been observed in accordance with the invention that either orboth of over-amplification of c-Myc mRNA and over-expression of C-MYCprotein in a tumor correlates with sensitivity of the tumor to treatmentwith agents that inhibit RNA polymerase II, including triptolide. Usingpatient-derived xenograft mouse models of pancreatic and ovarian cancerswith c-Myc gene amplification, sensitivity of tumors to triptolide wasobserved. In cases of high levels of c-Myc mRNA and high levels of c-MYCprotein, treatment with triptolide produced complete tumor eliminationand no regrowth in animals kept on a drug-free schedule from 30 to 60days.

It was further observed that these tumor cells produced high level ofnon-phosphorylated c-Myc protein that was prone for degradation by thetreatment with triptolide. It was observed that pancreatic cancer cellsthat carry multiple copies of the c-Myc gene and produce low or nophosphorylated c-MYC protein are ontogenically dependent on high levelsof c-Myc for their survival. The established triptolide-resistant PDXmodels of c-Myc amplified pancreatic cancer revealed increasedprotection of c-Myc protein by phosphorylation at Serine 62 residue andalso overexpression of ERCC3 protein, one of the established targets oftriptolide with critical role in the RNA polymerase II driventranscription process.

Multiple PDX models of pancreatic and ovarian cancers with no c-Mycamplification also showed sensitivity to triptolide that was associatedwith significant tumor shrinkage, which followed by immediate regrowthafter treatment cessation. Thus, it is believed that c-Myc amplificationand/or overexpression may serve as a biomarker to stratify patients withwide range of cancers by their enhanced sensitivity to the treatmentwith triptolide, its analogues and prodrugs, and also agents that targetRNA polymerase II driven transcription.

Accordingly, the invention features systems and methods for determiningwhether a cancer patient, especially an ovarian, pancreatic, gastric, oresophageal cancer patient, will respond positively to treatment withchemotherapeutic agents that inhibit the expression and/or biologicactivity of RNA polymerase II, including transcription mediated by RNApolymerase II. The invention alters the normal course of a treatmentmodality through RNA polymerase II inhibition by providing a checkpointinsofar as the information about c-Myc mRA and protein levels in a tumorpatient indicate whether or not that patient will appropriately respondto the RNA polymerase II inhibition therapy. In the case where thepatient will not appropriately respond to RNA polymerase II inhibitiontherapy, for example, because that patient's c-Myc mRNA and/or proteinlevels are insufficiently elevated, the patient can be administereddifferent treatment regimen that does not include RNA polymerase IIinhibition. Any of the methods may be carried out in vivo, in vitro, orin situ.

In some aspects, a system comprises a data structure, which comprisesreference values for levels of c-Myc mRNA, levels of c-MYC protein, orlevels of both c-Myc mRNA and c-MYC protein that correspond to thesensitivity of a tumor to treatment with an agent that inhibits thebiologic activity of RNA polymerase II. In tumors responsive to RNApolymerase II inhibition, levels of c-Myc mRNA and/or c-MYC protein areat least equal to, and in many cases are higher than a minimal thresholdfor RNA polymerase II inhibition to facilitate tumor cell death. Forexample, such a level may comprise a level that corresponds to the tumorbeing likely responsive to treatment with the agent (e.g., at or abovethe minimal threshold levels), and/or a level that corresponds to thetumor being likely unresponsive to treatment with the agent (e.g., belowthe minimal threshold levels). Responsiveness includes, for example,killing of cells in the tumor that come in contact with the agent. Thelevels of c-MYC protein may comprise levels for unphosphorylated c-MYC,and/or levels for c-MYC phosphorylated at the Serine 62 position.

The system also comprises a processor operably connected to the datastructure. The processor may comprise a computer processor. The systemmay comprise a computer network connection, for example, an Internetconnection. The processor may comprise various inputs and outputs.

Preferably, the processor is programmed to compare a level of c-MycmRNA, a level of c-MYC protein, or a level of both c-Myc mRNA and c-MYCprotein determined from a cancer patient with the reference valuesand/or levels of c-Myc mRNA, levels of c-Myc protein, or levels of bothc-Myc mRNA and c-MYC protein in the data structure, and is alsoprogrammed to determine whether the tumor in the patient is sensitive,including the degree of sensitivity, or not sensitive to treatment anagent that inhibits the biologic activity of RNA polymerase II. Forexample, the processor may be programmed to determine a RNA polymeraseII inhibition sensitivity score as a result of the comparison of thec-Myc mRNA and/or C-MYC protein levels in the patient with the referencevalue levels in the data structure. Thus, for example, once the level ofc-Myc mRNA and/or c-MYC protein in the patient's tumor cells isdetermined, the level may be entered into the system, and the level maythen be compared against the levels in the data structure, and if thepatient levels are high enough, a likelihood of responsiveness of thetumor to treatment with RNA polymerase II inhibitors can be determined.

The processor may determine a RNA polymerase II inhibitionsensitivity/response score based on the comparison of patient-samplec-Myc mRNA and/or C-MYC protein levels with the corresponding referencelevels in the data structure. The determined response score may then beprovided to a user, for example, a medical practitioner or the cancerpatient. Accordingly, in some aspects, the system optionally comprisesan output for providing the RNA polymerase II inhibitionsensitivity/response score to a user.

The form of the RNA polymerase II inhibition sensitivity/response scoreis not critical, and may vary according to the needs of the practitioneror user of the system. In its simplest form, such a response score maybe an indication whether the cancer patient, whose samples have beenentered into the system for comparison against the data structure, willor will not respond positively to chemotherapy that targets RNApolymerase II for inhibition. A positive response includes, for example,a clinically significant killing of tumor cells, including a reductionin the size of the solid tumor, and including elimination of the tumor.A positive response may also include, for example, stabilizing thecancer such that no further growth occurs. At least a partial positiveresponse may be considered a beneficial treatment outcome. A responsescore may comprise a scale of a likely positive response, for example, ascale of 1 to 10 or other suitable integers, with one end of thespectrum corresponding to a score that the patient likely will notrespond positively to RNA polymerase II inhibition chemotherapy and theother end of the spectrum corresponding to a score that the patientlikely will respond positively to RNA polymerase II inhibitionchemotherapy. A response score may comprise a value indicative of a highlikelihood of a positive response to RNA polymerase II inhibitionchemotherapy, a value indicative of a moderate likelihood of a positiveresponse to RNA polymerase II inhibition chemotherapy, or a valueindicative of a low likelihood of a positive response to RNA polymeraseII inhibition chemotherapy. In some aspects, a response score may bebacked up by statistical significance, according to any suitablestatistical methodology.

A response score may, for example, be a function of the level of c-MycmRNA and/or the level of c-MYC protein in the patient's tumor cells. Aresponse score may, for example, be a function of the type ofchemotherapy, including the particular chemotherapeutic agent(s) orcombinations thereof or dose thereof, or including the length oftreatment or route of administration, among other factors that accompanythe design and implementation of a particular chemotherapeutic regimenfor a given patient. The agent may comprise triptolide, an analog oftriptolide, a prodrug form of triptolide, or any combination thereof.

The particular agent(s) or combinations thereof preferably inhibit thebiologic activity of RNA polymerase II, including transcription mediatedby RNA polymerase II. In some aspects, the agent(s) or combinationsthereof induce degradation of RNA polymerase II. Degradation may resultfrom inhibition of the biologic activity, but degradation may also be amore direct function of the agent itself. RNA polymerase II degradationmay comprise proteasome degradation. For example, the agent(s) orcombinations thereof may induce proteasome degradation of RNA polymeraseII. Thus, in some aspects the RNA polymerase II inhibition sensitivityscore comprises a likelihood that the agent(s) or combinations thereofwill inhibit the biologic activity of RNA polymerase II in the tumor,thereby treating the tumor. In some aspects, the RNA polymerase IIinhibition sensitivity score comprises a likelihood that the agent(s) orcombinations thereof will induce degradation of RNA polymerase II in thetumor, thereby treating the tumor. In some aspects, the RNA polymeraseII inhibition sensitivity score comprises a likelihood that the agent(s)or combinations thereof will induce proteasome degradation of RNApolymerase II in the tumor, thereby treating the tumor.

In addition to or in the alternative to inhibiting the biologic activityof RNA polymerase II, the agent(s) or combinations thereof may inhibitthe expression or the biologic activity of the excision repaircross-complementing rodent repair deficiency, complementation group 3(ERCC3) protein. The agent(s) or combinations thereof may, for example,inhibit the transcription of the ERCC3 gene, or inhibit the translationof ERCC3 mRNA, or inhibit the biologic activity of the ERCC3 protein.Thus, in some aspects the RNA polymerase II inhibition sensitivity scorecomprises a likelihood that the agent(s) or combinations thereof willinhibit the biologic activity of ERCC3 in the tumor, including at thetranscription, translation, or protein activity level, thereby treatingthe tumor.

In addition to or in the alternative to inhibiting the biologic activityof RNA polymerase II, the agent(s) or combinations thereof may inhibitthe expression or the biologic activity of the c-MYC protein. Theagent(s) or combinations thereof may, for example, inhibit thetranscription of the c-Myc gene, or inhibit the translation of c-MycmRNA, or inhibit the biologic activity of the C-MYC protein. Thus, insome aspects the RNA polymerase II inhibition sensitivity scorecomprises a likelihood that the agent(s) or combinations thereof willinhibit the biologic activity of c-MYC in the tumor, including at thetranscription, translation, or protein activity level, thereby treatingthe tumor. In some aspects, agent(s) or combinations thereof may inhibitthe interaction of the ERCC3 protein with the C-MYC protein. In someaspects, agent(s) or combinations thereof may inhibit the KRAS-ERK-MYCsignal cascade.

In some aspects, the processor may be programmed to recommend aparticular treatment regimen for the patient, based on the RNApolymerase II inhibition sensitivity score. For example, the processormay recommend for patients who are determined to have a stronglikelihood of a positive response to be administered one or more agentsthat inhibit the biologic activity of RNA polymerase II. For example,the processor may recommend for patients who are determined to have astrong likelihood of a positive response to be administered a regimen oftriptolide, an analog of triptolide, a prodrug form of triptolide, orany combination thereof. The chemotherapeutic regimen may be directed bya medical practitioner according to patient care standards known orsuitable in the art.

Optionally, the system may comprise an input for entering c-Myc mRNAlevels and/or C-MYC protein levels determined or otherwise obtained frompatient samples into the system. Optionally, the system may comprise anoutput for providing results of a structure comparison, including aresponse score, to a user such as the subject, or a technician, or amedical practitioner.

The levels of c-Myc mRNA and/or levels of c-MYC protein in the datastructure may comprise levels for particular tumor types, preferably fortumors that respond to RNA polymerase II inhibition if the levels aresufficient. The tumors may be any tumors that overamplify and/oroverexpress c-MYC. The tumors may comprise pancreatic tumors. The tumorsmay comprise ovarian tumors. The tumors may comprise gastric tumors. Thetumors may comprise esophageal tumors.

In some aspects, the system may comprise computer readable mediacomprising executable code for causing a programmable processor tocompare a level of c-Myc mRNA and/or a level of c-MYC protein obtainedfrom a cancer patient with levels of c-Myc mRNA, and/or levels of c-MYCprotein that correspond to the sensitivity of a tumor to treatment withan agent that inhibits the biologic activity of RNA polymerase II, andfor causing a programmable processor to determine a RNA polymerase IIinhibition sensitivity score as a result of the comparison. Thesensitivity score may be as described above, including a likelihood thatthe cancer patient will or will not respond positively to chemotherapythat targets RNA polymerase II for inhibition. Such computer readablemedia are also featured in accordance with the invention separate fromthe systems of the invention. The computer readable media may comprise aprocessor, which may be a computer processor.

In one aspect, the invention provides methods for determining whether acancer patient may respond positively to inhibition of RNA polymeraseII. In some aspects, the methods generally comprise the steps ofcomparing a level of c-Myc mRNA and/or level of c-MYC protein from asample isolated from a cancer patient with levels of c-Myc mRNA and/orlevels of c-MYC protein that correspond to the sensitivity of a tumor totreatment with an agent that inhibits the biologic activity of RNApolymerase II, and determining whether the patient will respond tochemotherapy that inhibits RNA polymerase II based on the comparison.The cancer patient may be a pancreatic cancer patient. The cancerpatient may be an ovarian cancer patient. The sample isolated from thepatient may comprise a cell isolated from a tumor in the patient.

The comparing step may be carried out, for example, using a processorprogrammed to compare patient levels of c-Myc mRNA and/or c-MYC proteinwith reference values of levels of c-Myc mRNA and/or c-MYC protein thatcorrespond to the sensitivity of a tumor to treatment with an agent thatinhibits the biologic activity of RNA polymerase II. The referencevalues may, for example, be present in a data structure. The determiningstep may be carried out, for example, using a processor programmed todetermine whether a cancer patient will respond to RNA polymerase IIinhibition, based on the comparison of patient samples with referencevalues. In some aspects, determining whether the patient will respond toRNA polymerase II inhibition comprises generating a RNA polymerase IIinhibition sensitivity score as a result of the comparison. The responsescore may be as described above, including a likelihood that the patientwill (at least partially) or will not respond positively to RNApolymerase II inhibition. Inhibition may be via triptolide, an analog oftriptolide, a prodrug form of triptolide, or any combination thereof.

The systems, computer readable media, and platforms described orexemplified herein may be used in accordance with such methods. Forexample, the methods may comprise determining the level of c-Myc mRNA,the level of c-MYC protein, or the level of both c-Myc mRNA and c-MYCprotein from a sample, such as a tumor cell, isolated from the patient,entering the determined level into a system as described or exemplifiedherein, causing the processor of the system to compare the determinedlevel from the patient with a level of c-Myc mRNA, a level of c-MYCprotein, or a level of both c-Myc mRNA and c-MYC protein in the datastructure, with the data structure levels corresponding to a degree ofsensitivity of the tumor to treatment with an agent that inhibits thebiologic activity of RNA polymerase II, and causing the processor todetermine a RNA polymerase II inhibition sensitivity score based on thecomparison of patient and database levels of c-Myc mRNA and/or c-MYCprotein.

In some aspects, in which the cancer patient is determined to have alikelihood of responding positively to RNA polymerase II inhibitiontherapy, the methods may further comprise the steps of treating thepatient with an agent that inhibits the biologic activity of RNApolymerase II. Treating may include administering to the patient theagent in an amount effective to inhibit the biologic activity of RNApolymerase II.

The agent may comprise the plant Tripterygium wilfordii (the Thunder GodVine), or an extract thereof. The agent may comprise triptolide, ananalog of triptolide, a prodrug form of triptolide, or any combinationthereof. Triptolide may comprise the formula:

Triptolide analogs or prodrugs may comprise minnelide, or any moleculedescribed in U.S. Pat. Nos. 6,548,537, 6,150,539, or 7,863,464, or U.S.Publ. No. 2014/0107077, the entire contents of each of which areincorporated by reference herein. Administration may be according to anysuitable route.

Thus, the invention also features methods for treating a tumor in apatient in need thereof. In some aspects, the methods comprise comparingthe level of c-Myc mRNA and/or the level of c-MYC protein determinedfrom a tissue sample isolated from the patient, for example, a cell ortissue isolated from the tumor, with reference values for a level ofc-Myc mRNA and/or a level of c-MYC protein corresponding to a degree ofsensitivity of a tumor to treatment with an agent that inhibits thebiologic activity of RNA polymerase II and, based on the comparison, ifthe level of c-Myc mRNA and/or the level of c-MYC protein from thepatient is greater than or equal to a level of c-Myc mRNA, a level ofc-MYC protein, or a level of both c-Myc mRNA and c-MYC proteincorresponding to a degree of sensitivity of the tumor to treatment withan agent that inhibits the biologic activity of RNA polymerase II,contacting the tumor with an agent that inhibits the biologic activityof RNA polymerase II, thereby treating the tumor.

In some aspects, the methods comprise determining whether the level ofc-Myc mRNA and/or the level of c-MYC protein is greater than or equal toa level of c-Myc mRNA and/or a level of c-MYC protein corresponding to adegree of sensitivity of the tumor to treatment with an agent thatinhibits the biologic activity of RNA polymerase II and, based on thedetermination, if the level of c-Myc mRNA and/or the level of c-MYCprotein is greater than or equal to the level of c-Myc mRNA and/or thelevel of c-MYC protein corresponding to a degree of sensitivity of thetumor to treatment with an agent that inhibits the biologic activity ofRNA polymerase II, administering to the subject an agent that inhibitsthe biologic activity of RNA polymerase II in an amount effective toinhibit the biologic activity of RNA polymerase II, thereby treating thetumor.

In any of the methods, the tumor may comprise cells that over-amplifyc-Myc mRNA. The tumor may comprise cells that over-express the c-Mycprotein. The tumor may comprise a pancreatic tumor, such as a PDACtumor. The tumor may comprise an ovarian tumor. The tumor may comprise agastric tumor. The tumor may comprise an esophageal tumor.

Contacting the tumor may comprise administering to the patient the agentin an amount effective to inhibit the biologic activity of RNApolymerase II. Administration may be according to any suitableadministration modality, including, for example, oral administration orparenteral administration. The agent may inhibit the expression or thebiologic activity of the ERCC3 protein. The agent may inhibit thetranscription of the ERCC3 gene. The agent may inhibit the translationof ERCC3 mRNA. The agent may inhibit the biologic activity of the ERCC3protein. The agent may inhibit the transcription of the c-Myc gene. Theagent may inhibit the expression of the C-MYC protein. The agent mayinhibit the interaction of the ERCC3 protein with the C-MYC protein. Theagent may inhibit the KRAS-ERK-MYC signal cascade. The agent may inducedegradation of RNA polymerase II. The agent may comprise triptolide, ananalog of triptolide, a prodrug form of triptolide, or any combinationthereof. Administration may be at the direction of a medicalpractitioner.

The effective amount of the agent may be tailored to the level of c-Mycamplification and/or the level of c-MYC overexpression. For example, incases where c-Myc mRNA or c-MYC protein levels are high, less of the RNApolymerase II-inhibiting agent may be used, which may be particularlyhelpful for agents that have a high level of toxicity or a smalltherapeutic window. By way of example, by selecting patients with tumorsthat are particularly sensitive to triptolide, lower dosages oftriptolide may be administered, with a decrease in side effects andimprovement in the safety of the treatment.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1 Chemical Genomics Identified Transcription as a Target in PDAC

Through an IRB-approved protocol, patient-derived xenografts (PDX) andcell lines were derived from PDAC surgical samples. The technique ofrapid expansion (5-6 passages) avoided artificial selection of2D-adapted cell line subclones. Iterative chemosensitivity screens ofreprogrammed PDAC cells from 6 patients were conducted using a focusedlibrary of 867 drugs (NCI Clinical Collection and the FDA-approved drugset). This moderate scale, fully-automated screen assessed tumor cellviability at 6 drug concentrations ranging from 16 nM-10 μM. Sixteenhits were identified (area under the curve (AUC)<1.0, 50% inhibitoryconcentration (IC₅₀)

<100 nM) that were highly cytotoxic in these 6 cell lines. Ten of thesedrugs directly inhibit gene transcription, and none have been previouslyconsidered for PDAC therapy: triptolide was the most potent amongtranscriptional repressors including actinomycin D, epirubicin (andother anthracyclin antibiotics), plicamycin.

Example 2 Targeting ERCC3 with Triptolide Shows Unprecedented ActivityIn Vivo

The screen hits were then validated in vitro and, selected drugs weredirectly tested on a genetically-characterized panel of PDAC PDX models.Treatment of pancreatic PDX with a wide range of clinically availablecompounds typically produces temporary growth delays (FIG. 1A).Extensive testing of one of the PDAC patient-derived xenografts, PNX001,revealed transient growth delay during the 21-day treatment trials ofall tested agents, including a number of standard chemotherapies used inPDAC (FIG. 1A). It was determined that despite nearly identical growthrates of untreated patient-derived xenografts at F1-F3 passage (FIG.1B), there was a dramatic difference in their sensitivity to triptolide(FIG. 1C, 2A): Two models, YT037 (dark grey circles) and PNX001 (middlegrey circles), n=5 mice, 10 tumors in each group showed completeregression by the end of the 21-day treatment period. These miceremained tumor-free for at least 20 more days and exhibited no signs oftoxicity. Genotyping of the PDAC reprogrammed cells and PDX models byhigh resolution comparative genome hybridization (CGH), and RNA andexome sequencing showed that all sources of tumor cells from patientsPNX001 and Y1037 showed MYC amplification in addition to mutations inKRAS and TP53.

Example 3 ERCC3 as Target of Interest for PDAC

Such unprecedented efficacy of triptolide in MYC-amplified PDX modelsprompted further investigation of the drug's effect on itstranscriptional target, the RNA Pol II complex. Triptolide covalentlybinds to ERCC3, the xeroderma pigmen-tosum group B protein operating inthe leading edge of the megadalton RNA Pol II complex as a helicase anda DNA translocase critical for promoter opening and promoter escape. MYCmRNA is one of the shortest-lived and ranks as the primary target oftriptolide.

In both, sensitive PNX001 and refractory PNX007 cells (FIG. 2A),triptolide rapidly suppressed MYC transcripts (FIG. 2B). Contrastingly,triptolide-resistant PNX007 cells were insensitive to triptolide-inducedMYC degradation (FIG. 2C). Of note, phosphorylated MYC proteins (FIG.2C, pT58- and pS62-MYC) resisted triptolide effects in both cell lines.

Furthermore, pancreatic cancer PDX tumors that reappeared after completeregression (FIG. 3A), have acquired complete refractoriness totriptolide upon re-challenge. These tumors expressed markedly higherlevels of phosphoS62-MYC and ERCC3 (FIG. 3B).

The functional connection between MYC and ERCC3 is yet poorlyunderstood. It was first established that ERCC3 siRNA markedly decreasedMYC level on PNX001 cells (FIG. 4A). Conversely, MYC silencingdramatically depleted ERCC3 levels. This MYC-regulated expression ofERCC3 was further confirmed in P493-6 cells with Tet-repressor dependentexpression of MYC (FIG. 4B). Without intending to be limited to anyparticular theory or mechanism of action, it is believed that ERCC3blockade with siRNA or triptolide exerts its biological activity byderegulating short-lived mRNA including MYC.

These data suggest: 1) targeting transcription is highly active againstPDAC; 2) MYC is one of the primary targets of ERCC3/triptolide and thedriver of activated transcription in PDAC; 3) activity of KRAS-ERK-MYCaxis stabilizes MYC protein and confers resistance to transcriptionalinhibitors; hence, 4) combined targeting of transcription and KRAS-ERKpathway signaling may be synthetic lethal in PDAC.

Example 4 Basis for MYC-ERCC3 Interaction

The data (FIG. 3 and FIG. 4) show that ERCC3, as a critical part of RNAPol II-TFIIH complex, is essential for MYC-mediated transcription ofshort-lived transcripts for genes regulating cell cycle, apoptosis andtranscription itself. Multiple general transcriptional factors, but notERCC3, have been previously identified in RNAi screen as syntheticlethal with MYC, suggesting the dependency of MYC-driven cancers on hightranscription rate. It remains unclear, however, why ERCC3 was notidentified as a synthetic lethal target.

One possibility may be that triptolide uniquely inactivates ERCC3molecule rendering it a dominant negative protein, and thus moreeffectively disabling MYC-driven RNA Pol II. The data indicate thatincreased expression of ERCC3 promotes cell growth (FIG. 5), and confersresistance to triptolide in a MYC-dependent manner (FIG. 3 and FIG. 5).

Ongoing and future studies will directly address the possible dominantnegative mechanism for triptolide activity by overexpression of wildtype and catalytically inactive ERCC3 mutants. Whether the triptolidedifferential activity in PDAC cell lines is due to varying levels ofERCC3 expression will be tested. The full-length ERCC3 and severaltruncated forms of ERCC3, including a catalytically inactive K346R-ERCC3mutant, will be expressed to extend the experiments in HEK293 cells,(FIG. 5) in patient-derived early passage PDAC cells using lentiviralgene delivery (PNX001, Y1037, PNX007, PNX017). The growth,tumorogenicity, and triptolide chemosensitivity of these modified cellswill be determined. It is believed that the K346R-ERCC3 mutant mimicstriptolide via a dominant negative effect. Expression of this mutantwill be under the control of Tet-inducible promoter in the pHUSH vector.Using Gateway cloning and available panel of lentiviral expressionconstructs, a series of constitutive or Tet-regulated expressionvariants of ERCC3 will be generated in patient-derived pancreatic cancerlines. As an alternative approach, the levels of MYC or ERCC3 will bevaried with shRNA to determine: i) dependency of ERCC3 expression on MYClevels; and ii) sensitivity to triptolide in relationship to MYC andERCC3 expression.

Example 5 MYC Phosphorylation Via KRAS-MAPK Signaling as the Source ofResistance to Transcriptional Repressors

Preliminary results support the hypothesis that resistance totranscriptional repressors is mediated via MYC stabilization: 1)phosphorylated protein species of MYC are relatively insensitive toarrest of transcription with triptolide (which inactivates ERCC3, FIG.2C); 2) a subset of tumor cells that acquired resistance to triptolideexhibit highly elevated phospho-MYC and ERCC3 (FIG. 3); 3) MYCdestabilization by MEK inhibitors induces rapid compensatory responsefrom surface receptor signaling. Thus, it is believed that combinedtargeting of MYC with transcriptional repressors and destabilization ofMYC by blockade of signaling may be highly effective against PDAC.

The effect on sensitivity to triptolide and other transcriptioninhibitors will be tested by introduction of degradation-resistantT58A-MYC to PNX001 cells that are highly sensitive to triptolide andactinomycin D. Lentiviral delivery and Gateway cloning will be used asdescribed in Example 4. Expressing cells will be selected with puromycinor sorted by green fluorescence using GFP-containing pHUSH construct,and further validated by Western blotting. Cells induced to expressT58A-MYC by addition of tetracycline to culture medium will be testedfor sensitivity to triptolide and actinomycin D, or DNA-damagingchemotherapy (CPT11).

Example 6 Upstream Signaling Mechanisms Contributing to MYCPhosphorylation and Stabilization in Triptolide-Refractory PDAC Tumors

MYC actively antagonizes the expression of a range of surface oncogenicreceptor tyrosine kinases (RTK). MYC depletion releases this repressivemechanism and increases MAPK signaling which, in turn, stabilizes MYC,The RTK involved in activation of output from the KRAS-ERK-MYC axis inPDAC are yet unknown.

The patient-derived, tumor cell lines and triptoliderefractory PDX tumortissue will be used to identify these activated pathways. Solid phaseantibody arrays will be used.

Preliminary studies of triptolide-resistant PNX001 tumors demonstratedmarkedly increased FGFR1 expression in 2 out of 3 tumors tested (FIG.6A). Comparative proteome profiling of the triptolide-resistant andsensitive tumors derived from PNX001, YT037, and from PDX tissues thatexhibit primary resistance to triptolide (e.g. PNX007, PNX015) will beassessed. These PDX models have been already in vivo expanded, andcryopreserved for proteomics and re-implantation. It is believed thatresistance to triptolide and other transcription inhibitors will beassociated with accelerated signaling via RTK-KRAS pathway. Theseresults will serve as the mechanistic basis for combination strategiesaimed to disrupt MYC-stabilizing signaling.

Example 7 Inactivation of KRAS-ERK-MYC Signaling to Overcome Resistanceto Transcriptional Inhibitors in PDAC

Transcriptional repressors (triptolide, actinomycin D, anthracyclins)will be tested in combination with FGFR, RAF, MEK, (e.g., AZD6244,PD184352), ERK, PI3K, AKT inhibitors, DNA-damaging (CPT11, platinum),protein destabilizing drugs (HSP90 inhibitor STA9090), BCL2 familyinhibitors (ABT-199) in triptolide-refractory (PNX007, PNX015) andtriptolide-sensitive (PNX001, YT037) cell lines. The in vitro-expandedPDAC cells will be systematically probed for synergy of pair-wise drugcombinations (e.g., data in PNX001 cells with MEK and BCL inhibitors,Table 1, CI, coefficient of interaction). For statistical analysis,viability data will be formatted and processed in the R-package andassessed with the Chou-Talalay isobologram method (synergy defined asCl<<1). Synergistic combinations will be selected for future in vivotesting. In cases of drug-drug synergy, the effects of the combinationson MYC phosphorylation and stability will be assessed by Western blot.

TABLE 1 Drug (Molar Ratio) ED50 ED75 ED90 Average CI InterpretationTriptolide/MEKi 0.54 0.49 0.47 0.50 Strong (1:6200) SynergyTriptolide/BCLi 0.66 0.43 0.28 0.46 Strong (1:250) Synergy

Example 8 Targeting of KRAS-ERK-MYC Signaling and TranscriptionSynergism In Vivo in PDX Models of PDAC

It is desired to dismantle the KRAS-ERK-MYC axis in pancreatic cancerusing rationally designed drug synergies. As a proof-of-conceptexperiment, a clinically proven MEK inhibitor, selumetinib/AZD6244, wasselected to test for in vivo synergy with triptolide.

As the first step, the combination of triptolide and clinicallyavailable MEK inhibitor selumetinib/AZD6244 will be tested for synergismin vivo. Established triptolide-refractory PDX models will be used. Inbrief, 6-8 week old C.B17-SCID mice will be grafted in the flanks withcryopreserved PDAC tumor fragments soaked in Matrigel®. Animals (n=10per group) will be randomized to receive triptolide 0.2 mg/kg,selumetinib 100 mg/kg administered orally twice daily 5 days per weekfor 3 weeks, a combination of two drugs or saline. Tumor volumes will beassessed twice a week as (length×width2)/2 for 6 weeks, and animals willbe euthanized if tumor ulcerates or reaches 2000 mm³. Tumor volumedifferences among groups will be analyzed using generalized linearmodels assuming appropriate family and link functions. Where necessary,models will be estimated using generalized estimating equations toaccount for correlated data.

To capture changes in KRAS-ERK-MYC signaling and in the expression ofthe surface RTKs, a separate cohort of dedicated xenografts will becollected either untreated or when refractory tumors recur following theinitial response to triptolide (FIG. 3). Collected tumor tissues will beprocessed to obtain lysates (3 animals per group) and analyzed bycommercial protein array with further validation by Western blot.Additional studies will determine if targeting FGFR1 or other RTKs intriptolide-resistant tumors (FIG. 6) synergizes with transcriptionalrepressors.

The invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1-20. (canceled)
 21. A method for treating a subject having an ovariantumor or a gastric tumor, comprising detecting an overamplified level ofthe c-Myc gene, an overexpressed level of the c-MYC protein, or both anoveramplified level of the c-Myc gene and an overexpressed level of thec-MYC protein in a cell isolated from the tumor, determining that theserine at position 62 of the c-MYC protein is not phosphorylated, andadministering to the subject an effective amount of triptolide orminnelide.
 22. The method of claim 21, wherein the method comprisesdetecting an overamplified level of the c-Myc gene in the cell.
 23. Themethod of claim 21, wherein the method comprises detecting anoverexpressed level of the c-MYC protein in the cell.
 24. The method ofclaim 21, wherein the method comprises detecting both an overamplifiedlevel of the c-Myc gene in the cell and an overexpressed level of thec-MYC protein in the cell.
 25. The method of claim 21, whereintriptolide is administered to the subject.
 26. The method of claim 21,wherein minnelide is administered to the subject.
 27. The method ofclaim 21, further comprising administering to the subject an effectiveamount of selumetinib.
 28. The method of claim 21, wherein the subjecthas an ovarian tumor.
 29. The method of claim 21, wherein the subjecthas a gastric tumor.
 30. The method of claim 21, wherein the subject isa human.
 31. The method of claim 21, wherein triptolide is administeredorally to the subject.
 32. The method of claim 21, wherein triptolide isadministered parenterally to the subject.
 33. The method of claim 21,wherein minnelide is administered orally to the subject.
 34. The methodof claim 21, wherein minnelide is administered parenterally to thesubject.